The present invention relates to a novel method of designing xcex1-amylase mutants with predetermined properties, which method is based on the hitherto unknown three-dimensional structure of bacterial xcex1-amylases.
xcex1-Amylases (xcex1-1,4 glucan-4-glucanohydrolase, EC 3.2.1.1) constitute a group of enzymes which is capable of hydrolyzing starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. Almost all xcex1-amylases studied have a few conserved regions with approximately the same length and spacing. One of these regions resembles the Ca2+ binding site of calmodulin and the others are thought to be necessary for the active centre and/or binding of the substrate.
While the amino acid sequence and thus primary structure of a large number of xcex1-amylases are known, it has proved very difficult to determine the three-dimensional structure of all xcex1-amylases. The three-dimensional structure can be determined by X-ray crystallographic analysis of xcex1-amylase crystals, but it has proven difficult to obtain xcex1-amylase crystals suitable for actually solving the structure.
Until now the three-dimensional structure of only a few xcex1-amylases-have been determined at high resolution. These include the structure of the Aspergillus oryzae TAKA xcex1-amylase (Swift et al., 1991), the Aspergillus niger acid amylase (Brady et al, 1991), the structure of pig pancreatic xcex1-amylase (Qian et al., 1993), and the barley alpha-amylase (Kadziola et al. 1994, Journal of Molecular Biology 239: 104-121, A.Kadziola, Thesis, Dept of Chemistry, U. of Copenhagen, Denmark). Furthermore, the three-dimensional structure of a Bacillus circulans cyclodextrin glycosyltransferase (CGTase) is known (Klein et al., 1992) (Lawson et al., 1994). The CGTase catalyzes the same type of reactions as xcex1-amylases and exhibits some structural resemblance with xcex1-amylases.
Furthermore, crystallization and preliminary X-ray studies of B. subtilis xcex1-amylases have been described (Chang et al. (1992) and Mizuno et al. (1993)). No final B. subtilis structure has been reported. Analogously, the preparation of B. licheniformis xcex1-amylase crystals has been reported (Suzuki et al. (1990), but no subsequent report on X-ray crystallographic analysis or three-dimensional structure are available.
Several research teams have attempted to build three-dimensional structures on the basis of the above known xcex1-amylase structures. For instance, Vihinen et al. (J. Biochem. 107, 267-272, 1990), disclose the modelling (or computer simulation) of a three-dimensional structure of the Bacillus stearothermophilus xcex1-amylase on the basis of the TAKA amylase structure. The model was used to investigate hypothetical structural consequences of various site-directed mutations of the B. stearothermophilus xcex1-amylase. E. A. MacGregor (1987) predicts the presence of xcex1-helices and 3-barrels in xcex1-amylases from different sources, including barley, pig pancreas and Bacillus amyloliquefaciens on the basis of the known structure of the A. oryzae TAKA xcex1-amylase and secondary structure predicting algorithms. Furthermore, the possible loops and subsites which may be found to be present in, e.g., the B. amyloliquefaciens xcex1-amylase are predicted (based on a comparison with the A. oryzae sequence and structure).
A. E. MacGregor (Starch/Stxc3xa4rke 45 (1993), No. 7, p. 232-237) presents a review of the relationship between the structure and activity of xcex1-amylase related enzymes.
Hitherto, no three-dimensional structure has been available for the industrially important Bacillus xcex1-amylases (which in the present context are termed xe2x80x9cTermamyl-like xcex1-amylasesxe2x80x9d), including the B. licheniformis, the B. amyloliquefaciens, and the B. stearothermophilus xcex1-amylase.
The three-dimensional structure of a Termamyl-like bacterial xcex1-amylase has now been elucidated. On the basis of an analysis of said structure it is possible to identify structural parts or specific amino acid residues which from structural or functional considerations appear to be important for conferring the various properties to the Termamyl-like xcex1-amylases. Furthermore, when comparing the Termamyl-like xcex1-amylase structure with known structures of the fungal and mammalian xcex1-amylases mentioned above, it has been found that some similarities exist between the structures, but also that some striking, and not previously predicted structural differences between the xcex1-amylases exist. The present invention is based on these findings.
Accordingly, in a first aspect the invention relates to a method of constructing a variant of a parent Termamyl-like xcex1-amylase, which variant has xcex1-amylase activity and at least one altered property as compared to said parent xcex1-amylase, which method comprises
i) analysing the structure of the Termamyl-like xcex1-amylase with a view to identifying at least one amino acid residue or at least one structural part of the Termamyl-like xcex1-amylase structure, which amino acid residue or structural part is believed to be of relevance for altering said property of the parent Termamyl-like xcex1-amylase (as evaluated on the basis of structural or functional considerations),
ii) constructing a Termamyl-like xcex1-amylase variant, which as compared to the parent Termamyl-like xcex1-amylase, has been modified in the amino acid residue or structural part identified in i) so as to alter said property, and, optionally,
iii) testing the resulting Termamyl-like xcex1-amylase variant with respect to said property.
In a second aspect the present invention relates to a method of constructing a variant of a parent Termamyl-like xcex1-amylase, which variant has xcex1-amylase activity and one or more altered properties as compared to said parent xcex1-amylase, which method comprises
i) comparing the three-dimensional structure of the Termamyl-like xcex1-amylase with the structure of a non-Termamyl-like xcex1-amylase,
ii) identifying a part of the Termamyl-like xcex1-amylase structure which is different from the non-Termamyl-like xcex1-amylase structure,
iii) modifying the part of the Termamyl-like xcex1-amylase identified in ii) whereby a Termamyl-like xcex1-amylase variant is obtained, one or more properties of which differ from the parent Termamyl-like xcex1-amylase, and optionally,
iv) testing the resulting Termamyl-like xcex1-amylase variant with respect to said property or properties.
In a third aspect the invention relates to a method of constructing a variant of a parent non-Termamyl-like xcex1-amylase, which variant has xcex1-amylase activity and one or more altered properties as compared to said parent xcex1-amylase, which method comprises
i) comparing the three-dimensional structure of the non-Termamyl-like xcex1-amylase with the structure of a Termamyl-like xcex1-amylase,
ii) identifying a part of the non-Termamyl-like xcex1-amylase structure which is different from the Termamyl-like xcex1-amylase structure,
iii) modifying the part of the non-Termamyl-like xcex1-amylase identified in ii) whereby a non-Termamyl-like xcex1-amylase variant is obtained, one or more properties of which differ from the parent non-Termamyl-like xcex1-amylase, and optionally,
iv) testing the resulting non-Termamyl-like xcex1-amylase variant with respect to said property or properties.
The property which may be altered by the above methods of the present invention may, e.g., be substrate specificity, substrate binding, substrate cleavage pattern, temperature stability, pH dependent activity, pH dependent stability (especially increased stability at low (e.g. pH less than 6, in particular pH less than 5) or high (e.g. pH greater than 9) pH values), stability towards oxidation, Ca2+-dependency, specific activity, and other properties of interest. For instance, the alteration may result in a variant which, as compared to the parent Termamyl-like xcex1-amylase, has an increased specific activity at a given pH and/or an altered substrate specificity.
In still further aspects the invention relates to variants of a Termamyl-like xcex1-amylase, DNA encoding such variants and methods of preparing the variants. Finally, the invention relates to the use of the variants for various industrial purposes.